Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/20—Milk; Whey; Colostrum
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/142—Amino acids; Derivatives thereof
  • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K50/00—Feeding-stuffs specially adapted for particular animals
  • A23K50/40—Feeding-stuffs specially adapted for particular animals for carnivorous animals, e.g. cats or dogs
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
  • A23L2/52—Adding ingredients
  • A23L2/66—Proteins
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/17—Amino acids, peptides or proteins
  • A23L33/18—Peptides; Protein hydrolysates
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
  • A23P10/00—Shaping or working of foodstuffs characterised by the products
  • A23P10/20—Agglomerating; Granulating; Tabletting
  • A23P10/28—Tabletting; Making food bars by compression of a dry powdered mixture
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/04—Anorexiants; Antiobesity agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/06—Antihyperlipidemics

Definitions

• a reagent that has a superior ability to improve lipid metabolism, is less bitter, has excellent stability, and is safe.
• This reagent can inhibit human and other mammalian adipocytes from incorporating lipids.
• This application will also describe food, beverages, nutritional supplement, and fodder that comprise this reagent.
• the second method involves the inhibition of fat accumulation, i.e., prevention of obesity.
• the two methods are considered to be the same, the actual mechanism behind them differs.
• the latter method which inhibits fat accumulation, has received attention for the prevention and treatment of obesity from the aspect of health and beauty.
• Attempted therapeutic methods include drug and exercise therapy and diet restrictions. Although drug therapy is expected to be effective, there is also a need to consider their side effects. Exercise therapy and diet restrictions accompany temporal or psychological difficulties in terms of compliance in the long term and have low success rates.
• soybean peptides from soybean proteins are known to inhibit accumulation of these indices (refer to NON PATENT LITERATURE 1).
• hydrolysate from whey protein has been demonstrated to possess lipid metabolism improving ability (refer to PATENT LITERATURE 3).
• Hydrolysate from milk proteins is used in various products to prevent food allergies from milk and dairy products.
• Whey protein obtained from cow milk differs from that of breast milk and is believed to be an allergen.
• enzymatic hydrolysis of whey protein, its method of preparation (refer to PATENT LITERATURE 4 and 5), and the method of denaturation of whey protein by heat and enzymatic hydrolysis by a heat-resistant hydrolase under specific conditions to obtain whey protein hydrolysate has been demonstrated (refer to Patent literature 6).
• Whey protein hydrolysate from milk is advantageous in that there is a higher content of branched amino acids compared with soy peptides from soy proteins (PATENT LITERATURE 2), which can produce a better lipid metabolism-improving reagent.
• a typical whey protein hydrolysate solution is turbid, has limitations when used as an actual product, and is incapable for use as products that are required to be transparent in appearance.
• peptides have a characteristic bitter flavor, limiting their use in orally consumed food and fodders.
• the objective of this invention is to produce a lipid metabolism-improving reagent or food, beverage, nutritional supplement, or fodder that contains such reagents and can be consumed as a part of the normal diet.
• their application in improving lipid metabolism and preventing or treating metabolic syndromes is not limited, because it is very safe and the aqueous solution is highly transparent and minimally bitter in flavor.
• Lipid metabolism-improving reagent comprising whey protein hydrolysate as the active ingredient with the following criteria:
• APL Average peptide chain length
• the lipid metabolism-improving reagent according to item (1) above characterized in that the whey protein hydrolysate can be obtained by denaturation of whey protein at pH 6-10 and 50° C.-70° C. and enzymatic hydrolysis by a heat-resistant hydrolase, followed by further heating to inactivate the enzyme.
• the lipid metabolism-improving reagent according to item (1) above characterized in that the whey protein hydrolysate can be obtained by the enzymatic hydrolysis of whey protein at pH 6-10 and 20° C.-55° C., followed by heating at pH 6-10 and 50° C.-70° C. for enzymatic hydrolysis of the unhydrolyzed whey protein with a heat-resistant hydrolase while being denatured by heat and further heating to inactivate the enzyme.
• Nutritional supplements comprising the lipid metabolism-improving reagents according to any one of items (1)-(3) above.
• Fodders comprising the lipid metabolism-improving reagents according to any one of items (1)-(3) above.
• APL Average peptide chain length
• whey protein hydrolysate can be obtained by denaturation of whey protein at pH 6-10 and 50° C.-70° C. and enzymatic hydrolysis by a heat-resistant hydrolase, followed by further heating to inactivate the enzyme.
• whey protein hydrolysate can be obtained by the enzymatic hydrolysis of whey protein at pH 6-10 and 20° C.-55° C., followed by heating at pH 6-10 and 50° C.-70° C. for enzymatic hydrolysis of the unhydrolyzed whey protein with a heat-resistant hydrolase while being denatured by heat and further heating to inactivate the enzyme.
• APL Average peptide chain length
• whey protein hydrolysate can be obtained by denaturation of whey protein at pH 6-10 and 50° C.-70° C. and enzymatic hydrolysis by a heat-resistant hydrolase, followed by further heating to inactivate the enzyme.
• whey protein hydrolysate can be obtained by the enzymatic hydrolysis of whey protein at pH 6-10 and 20° C.-55° C., followed by heating at pH 6-10 and 50° C.-70° C. for enzymatic hydrolysis of the unhydrolyzed whey protein with a heat-resistant hydrolase while being denatured by heat and further heating to inactivate the enzyme.
• APL Average peptide chain length
• whey protein hydrolysate can be obtained by denaturation of whey protein at pH 6-10 and 50° C.-70° C. and enzymatic hydrolysis by a heat-resistant hydrolase, followed by further heating to inactivate the enzyme.
• whey protein hydrolysate can be obtained by the enzymatic hydrolysis of whey protein at pH 6-10 and 20° C.-55° C., followed by heating at pH 6-10 and 50° C.-70° C. for enzymatic hydrolysis of the unhydrolyzed whey protein with a heat-resistant hydrolase while being denatured by heat and further heating to inactivate the enzyme.
• APL Average peptide chain length
• (K) The method according to item (J) above characterized in that the whey protein hydrolysate can be obtained by denaturation of whey protein at pH 6-10 and 50° C.-70° C. and enzymatic hydrolysis by a heat-resistant hydrolase, followed by further heating to inactivate the enzyme.
• whey protein hydrolysate can be obtained by the enzymatic hydrolysis of whey protein at pH 6-10 and 20° C.-55° C., followed by heating at pH 6-10 and 50° C.-70° C. for enzymatic hydrolysis of the unhydrolyzed whey protein with a heat-resistant hydrolase while being denatured by heat and further heating to inactivate the enzyme.
• This lipid metabolism-improving reagent has marked inhibitory effects on triglyceride and total cholesterol accumulation in serum and triglyceride accumulation in the liver. It is effective in the prevention and treatment of diseases such as hyperlipidemia and obesity.
• the whey protein hydrolysate mixed with the lipid metabolism-improving reagent can improve lipid metabolism.
• the hydrolysate can be obtained by denaturing the whey protein at 50° C.-70° C. and pH 6-10 and enzymatic hydrolysis by a heat-resistant hydrolase, followed by further heating to inactivate the enzyme. Prior to performing the above enzymatic hydrolysis, if the whey protein is hydrolyzed by a protein hydrolase at pH 6-10 and 20-55° C. and is not cooled, the yield is considerably higher.
• the molecular weight cutoff of the ultrafiltration membrane should be in the range of 1-20 kDa, or preferably 2-10 kDa.
• Molecular weight cutoff of the microfiltration membrane should be in the range of 100-500 Da, or preferably 150-300 Da.
• Transparency test (described below) should result in an absorbance below 0.014, preferably less than 0.010 or less than 0.005.
• the whey protein in this invention was an aggregate, powder, or purified protein derived from the whey of milks obtained from different mammals, such as cows, buffaloes, goats, or humans.
• the whey protein enzyme reaction was conducted in aqueous solution conditions.
• the pH of the whey protein aqueous solution is usually 6-10; therefore, there is no need to adjust the pH when enzymatic reactions occur.
• acidic solution such as hydrochloric acid, citric acid, or lactic acid or alkaline solution, such as sodium hydroxide, calcium hydroxide, or sodium phosphate, can be used to adjust the pH accordingly (pH6-10).
• this invention described heating at 50-70° C., it is preferable to add the heat-resistant hydrolase prior to heating the whey protein solution and carry out the enzymatic hydrolysis to obtain a higher yield.
• the optimal temperature for the heat-resistant hydrolase used in this method was ⁇ 45° C.
• any enzyme can be used without restrictions.
• Some examples of heat-resistant hydrolases are papain, protease S (brand name), proleather (brand name), thermoase (brand name), alcalase (brand name), and protin-A (brand name).
• a heat-resistant hydrolase that has about 10% or more residual activity after heating for 30 min at 80° C. is desired.
• use of multiple enzymes is more effective.
• the preferred reaction time is 30 min-10 h.
• reaction solution needs to be heated to inactivate the hydrolase.
• the enzyme that was used in this method can be inactivated by heating the reaction solution for 10 s or more at 100° C. and above.
• reaction solution collected as mentioned above was centrifuged and the supernatant was collected.
• the supernatant is dried to obtain the powdered whey protein hydrolysate product.
• the precipitate formed during centrifugation has less effect on decreasing allergic reactions compared with that of the supernatant and this should be removed.
• the reaction solution can be dried and used as it is.
• the whey protein hydrolysate which can be obtained by this method is quantitated by inhibition ELISA [Japanese Journal of Pediatric Allergy 1, 36 (1987)]. Its antigenicity was confirmed to be 1/10,000 or less compared with that of ⁇ -lactoglobulin and ⁇ 1/10,000 of whey protein and is extremely safe. In addition, because the whey protein hydrolysate solution is transparent, and its bitterness score is approximately 2, there are no limitations to the use of this product. The transparency and the bitterness were assessed by the following methods.
• Transparency assessment method One percent whey protein hydrolysate solution was prepared and absorbance was measured at 650 nm.
• the whey protein hydrolysate can be directly used as a lipid metabolism-improving reagent, it can also be used in powder, granule, tablet, capsule, or solution form in the usual method.
• Whey protein hydrolysates which are processed by ultrafiltration or microfiltration, can be directly used as lipid metabolism-improving reagent or they can be used in dried form. In addition, it can be converted into various forms using the usual method.
• the food and beverages, nutritional supplements, and fodder of the present invention which have lipid metabolism improving effect may mean the whey protein hydrolysate itself. Moreover, they can include a normal constituent, such as stabilizers, sugars, flavors, vitamins, and minerals, flavonoids, and polyphenols.
• the food and beverages, nutritional supplements, and fodder of the present invention may be prepared by combining with other commonly used raw materials.
• whey protein hydrolysate there are no limitations to the use of whey protein hydrolysate in food, beverages, nutritional supplements, and fodders.
• its composition should be 0.001%-10% (w/w) or preferably 0.1%-5% (w/w) of the total mass, depending on the formulation.
• Appropriate additives can be added to the active ingredient to produce a preferred formulation of the lipid metabolism-improving reagent and this preparation can be prepared as an oral or non-oral preparation.
• diluents and excipients such as commonly used filler reagents, bulking reagents, bonding and disintegrating agents, surfactants, and lubricants can be used.
• Formulation types including capsules, tablets, granules, powders, solution, suspension, emulsion, suppositories, injections, and ointments, can be used.
• excipients include sucrose, lactose, starch, microcrystalline cellulose, mannitol, light anhydrous silicic acid, magnesium aluminate, synthetic aluminium silicate, magnesium, metasilicate aluminate, calcium carbonate, sodium bicarbonate, dibasic calcium phosphate, and carmellose calcium. Two or more of these can be mixed as an additive as well.
• this aqueous solution has unlimited use as a lipid metabolism-improving reagent, particularly as lipid metabolism-improving reagent that are required to be transparent in appearance.
• antigenicity was quantitated to be 1/10,000 or less compared with that of ⁇ -lactoglobulin with a yield of 80.3% (after centrifugation of the fluid in which the enzyme reaction occurred, yield was calculated by dividing the dry weight of the total reaction mixture by the dry weight of the supernatant) and bitterness score of 2.
• the whey protein hydrolysate obtained in this manner can be directly used as a lipid metabolism-improving reagent.
• the molecular weight distribution of the obtained whey protein hydrolysate was 10 kDa or less with main peak of 500 Da, APL of 3.0, and free amino acid content of 15.2% in total composition.
• Inhibition ELISA showed an antigenicity of 1/10,000 or less than that of ⁇ -lactoglobulin, a yield of 65.4%, and bitterness score of 2.
• the whey protein hydrolysate obtained using this method can be directly used as a lipid metabolism-improving reagent.
• the serum cholesterol, triglyceride, and phospholipid levels were measured using the commercial enzyme kits: Cholesterol E-test Wako, Triglyceride E-test Wako, and Phospholipid C-test Wako. All data are shown as mean ⁇ standard error (SE). Statistical analysis was performed using the Tukey-Kramer multiple comparison test and a P ⁇ 0.05 was considered statistically significant.
• TABLE 3 shows the values of body, organ, adipose tissue weights, and food consumption.
• Kidney weight was measured per 100 g of body weight and showed a significantly large value in the whey protein hydrolysate group compared with that of the control group. On comparing the group with respect to the heart and lung weights (per 100 g of body weight), no major difference was observed.
• Serum cholesterol level was lower (approximately 20%) in the soy peptide and whey protein hydrolysate groups compared with that of the control group.
• Serum phospholipid and triglyceride concentrations had similar results, and the whey protein hydrolysate group showed a smaller value compared with that of the soy peptide group.
• whey protein hydrolysate solution is transparent and its bitterness score is as low as 2; therefore, there is no limitation in its use in manufactured products. Thus, there is a clear effect of suppressing accumulation of serum triglyceride, cholesterol, and phospholipid.
• whey protein hydrolysate obtained from EXAMPLE 2 was dissolved, heated to 50° C., and mixed for 30 min at 6000 rpm using a TK homomixer (TK ROBO MICS; Tokushu Kika Kogyo) to obtain a whey protein hydrolysate solution with a whey protein concentration of 50 g/5 kg.
• TK homomixer TK ROBO MICS; Tokushu Kika Kogyo
• the following ingredients were added to 5.0 kg of the whey protein hydrolysate solution: 5.0 kg of casein, 5.0 kg of soy protein, 1.0 kg of fish oil, 3.0 kg of perilla oil, 18.0 kg of dextrin, 6.0 kg of minerals, 1.95 kg of vitamins, 2.0 kg of emulsifier, 4.0 kg of stabilizer, and 0.05 kg of flavor.
• This mixture was filled into a 200 mL retort pouch and sterilized for 20 min at 121° C. with a retort sterilizer (First pressure vessel; TYPE: RCS-4CRTGN; Hisaka works) to produce 50 kg of nutritional supplement.
• the amount of whey protein hydrolysate in 100 g of the nutritional supplement was 100 mg.
• the amount of whey protein hydrolysate in 100 ml of this beverage was 100 g.
• whey protein hydrolysate solution in 99.8 kg of deionized water, 200 g of the whey protein hydrolysate obtained from EXAMPLE 2 was dissolved, heated to 50° C., and stirred for 40 min at 3600 rpm using TK homomixer (MARK II 160 type: Tokushu Kika Kogyo) to obtain whey protein hydrolysate solution with a whey protein concentration of 2 g/100 g.
• TK homomixer MARK II 160 type: Tokushu Kika Kogyo
• whey protein hydrolysate solution 12 kg of soy bean flour (soybean meal), 14 kg of skim milk, 4 kg of soy oil, 2 kg of corn oil, 23.2 kg of palm oil, 14 kg of corn starch, 9 kg of wheat flour, 2 kg of wheat bran, 5 kg of vitamins, 2.8 kg of cellulose, and 2 kg of minerals.
• soy bean flour soybean meal
• skim milk 4 kg of soy oil
• 2 kg of corn oil 23.2 kg of palm oil
• 14 kg of corn starch 9 kg of wheat flour
• 2 kg of wheat bran 5 kg of vitamins, 2.8 kg of cellulose, and 2 kg of minerals.
• the amount of whey protein hydrolysate in 100 g of dog food was 20 mg.

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• 2009
  • 2009-04-16 JP JP2009100147A patent/JP5735734B2/ja active Active
• 2010
  • 2010-04-08 AU AU2010237964A patent/AU2010237964B2/en active Active
  • 2010-04-08 CN CN2010800168144A patent/CN102395376A/zh active Pending
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  • 2010-04-08 WO PCT/JP2010/056349 patent/WO2010119803A1/ja active Application Filing
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